Three new metabolites of hybrid strain KO 0231, derived from Penicillium citreo-viride IFO 6200 and 4692

Article abstract of DOI:10.1016/S0040-4020(00)00157-5

Three new metabolites, anthrasteroids, have been isolated from the mycelia of a hybrid strain, KO 0231, prepared by cell fusion technique using Penicillium citreo-viride IFO 6200 and 4692. Their structures were determined based on their spectral data. Fur

Full text of DOI:10.1016/S0040-4020(00)00157-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2595±2602
Three New Metabolites of Hybrid Strain KO 0231, Derived from
Penicillium citreo-viride IFO 6200 and 4692
*
Takashi Nakada and Shosuke Yamamura
Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama 223-8522, Japan
Received 6 January 2000; accepted 16 February 2000
AbstractÐThree new metabolites, anthrasteroids, have been isolated from the mycelia of a hybrid strain, KO 0231, prepared by cell fusion
technique using Penicillium citreo-viride IFO 6200 and 4692. Their structures were determined based on their spectral data. Furthermore,
these natural products were synthesized starting from ergosterol. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
bioactive compounds. We describe herein another
example using a hybrid strain, KO 0231, prepared from P.
citreo-viride IFO 6200 and 4692.
In previous papers, a variety of bioactive metabolites with
novel structures have been found in the mycelia of hybrid
strains prepared by cell fusion of two different strains, Peni-
cillium citreo-viride IFO 6200 and 4692. Of the two parent
strains, the former mainly produced citreoviridin (1)1,2 and
related a-pyrones,3,4 while the latter, P. citreo-viride IFO
4692, produced many phenolic compounds represented by
citreoviranol (2).5 Interestingly, however, the representative
metabolites of hybrid strains have novel structures different
from those of the two parents: citreofuran (3),6 citreohybri-
done A (4),7,8 citreothiopyrane A (5)9 and citreospirosteroid
(6)10 are produced by the hybrid strains ME 0005, KO 0031,
KO 0201 and KO 0011, respectively (Scheme 1). Therefore,
the cell fusion technique is quite useful for ®nding new
Results and Discussion
According to the usual procedures previously reported, we
were able to isolate three new metabolites (I, II and III)
from the mycelium of a hybrid strain, KO 0231, newly
prepared by cell fusion experiments using P. citreo-viride
IFO 6200 and 4692 (Scheme 2). Their structures were deter-
1
mined based on the H and 13C NMR, HMQC and HMBC
spectra with the aid of NOE experiments.
Citreoanthrasteroid A (I) as a colorless oil has a molecular
Scheme 1.
Keywords: hybrid strain; cell fusion; anthrasteroids.
* Corresponding author. Tel.: 11-81-45-563-5967; fax: 11-81-45-563-5967; e-mail: yamamura@chem.keio.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00157-5

2596
T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
Scheme 2.
formula C28H40O2 and one unsaturated CO and sec-OH
group each (CO: IR 1667 cm21 and 13C NMR d
including aromatic ring with one methyl group C-19 (1H
and 13C NMR d 2.47 and 16.4 ppm) at C-10; C-11±C-17
with one methyl group C-18 (1H and 13C NMR d 0.60 and
13.2 ppm) at C-13; C-20±C-28 with four methyls and the
above-mentioned ole®nic double bond], as shown in Fig. 1.
1
201.2 ppm; OH: IR 3397 cm21, H and 13C NMR d 4.19
and 67.4 ppm). The 1H NMR spectrum indicates the
presence of a disubstituted trans-ole®nic double bond (d
1
5.27 and 5.23 ppm), as shown in Table 1, where the H
and 13C NMR spectral data were unambiguously assignable
to each proton and carbon based on proton homonuclear
decoupling and HMBC experiments. The partial structure
(C-1±C-4) including the sec-OH group was determined by
proton homonuclear decoupling experiments. The remain-
ing moieties were also determined based on proton homo-
nuclear decoupling and HMBC experiments [C-5±C-10
Citreoanthrasteroid B (II) as a colorless oil has a molecular
1
formula C28H40O. The H and 13C NMR spectra of II are
quite similar to those of citreoanthrasteroid A (I) except for
1
the following points, as shown in Table 1. In the H NMR
spectrum, II has a cis-ole®nic double bond [d 6.48 ppm
(1H, d, Jˆ9.9 Hz) and d 6.57 ppm (1H, d, Jˆ9.9 Hz)],
while there is the ±CO±CH2± grouping [d 201.2 ppm
Table 1. 1H and 13C NMR data of anthrasteroids
C no.
Citreoanthrasteroid A (I)
Citreoanthrasteroid B (II)
Secocitreoanthrasteroid (III)
13C
27.8
30.4
1H
13C
28.0
31.3
1H
13C
27.5
31.2
1H
1
2
2.80 (1H, m)
2.95 (1H, dt, Jˆ17.6, 5.9 Hz)
1.77 (1H, m)
2.85 (1H, m)
2.79 (2H, m)
1.72 (1H, m)
2.09 (1H, m)
4.16 (1H, m)
1.72 (1H, m)
2.02 (1H, m)
4.10 (1H, m)
2.04 (1H, m)
4.19 (1H, m)
3
4
67.4
36.1
68.1
36.6
68.2
36.4
2.57 (1H, dd, Jˆ6.8, 16.6 Hz)
2.52 (1H, dd, Jˆ7.7, 16.3 Hz)
2.50 (1H, m)
2.95 (1H, dd, Jˆ5.5, 16.1 Hz)
3.04 (1H, dd, Jˆ5.4, 16.6 Hz)
3.04 (1H, dd, Jˆ5.1, 16.3 Hz)
5
6
7
8
129.1
140.7
124.1
142.7
132.2
139.8
201.2
56.9
130.0
135.1
123.4
137.1
130.7
131.7
123.0
139.9
124.8
136.2
113.4
152.3
124.5
135.3
22.2
6.71 (1H, s)
6.64 (1H, s)
6.51 (1H, s)
9
10
11
12
6.48 (1H, d, Jˆ9.9 Hz)
6.57 (1H, d, Jˆ9.9 Hz)
2.75 (2H, m)
1.72 (2H, m)
2.51 (1H, d, Jˆ17.9 Hz)
2.87 (1H, d, Jˆ17.9 Hz)
36.1
13
14
15
44.4
50.7
28.7
68.1
50.2
29.6
52.5
226.2
36.9
2.83 (1H, m)
2.08 (2H, m)
2.85 (1H, m)
1.60 (1H, m)
2.05 (1H, m)
1.70 (1H, m)
1.90 (1H, m)
1.65 (1H, m)
0.58 (3H, s)
2.18 (3H, s)
2.05 (1H, m)
2.10 (1H, m)
2.45 (1H, m)
2.05 (1H, m)
1.50 (1H, m)
1.92 (1H, m)
0.93 (3H, s)
2.12 (3H, s)
2.22 (1H, m)
16
24.0
1.95 (2H, m)
22.0
24.2
17
18
19
20
21
22
23
24
25
26
27
28
55.9
13.2
16.4
40.4
20.6
132.6
134.9
42.8
33.1
19.6
19.9
17.6
1.62 (1H, dq, Jˆ11.7, 5.9 Hz)
0.60 (3H, s)
2.47 (3H, s)
51.7
11.6
14.6
41.0
20.9
132.3
135.1
43.6
33.2
19.8
20.1
17.7
46.5
17.6
15.6
39.2
20.7
133.6
134.1
42.9
33.1
19.7
20. 0
17.6
2.00 (1H, m)
1.02 (3H, d, Jˆ6.8 Hz)
5.21 (1H, dd, Jˆ7.3, 15.2 Hz)
5.23 (1H, dd, Jˆ8.4, 15.2 Hz)
1.86 (1H, q, Jˆ6.8 Hz)
1.48 (1H, m)
1.13 (3H, d, Jˆ6.8 Hz)
5.21 (1H, dd, Jˆ5.9, 15.0 Hz)
5.24 (1H, dd, Jˆ6.6, 15.0 Hz)
1.85 (1H, m)
1.17 (3H, d, Jˆ7.0 Hz)
5.33 (1H, dd, Jˆ7.3, 15.2 Hz)
5.25 (1H, dd, Jˆ8.4, 15.2 Hz)
1.91 (1H, m)
1.50 (1H, m)
1.50 (1H, m)
0.81 (3H, d, Jˆ6.8 Hz)
0.83 (3H, d, Jˆ6.4 Hz)
0.91 (3H, d, Jˆ6.8 Hz)
0.82 (3H, d, Jˆ6.2 Hz)
0.84 (3H, d, Jˆ6.6 Hz)
0.92 (3H, d, Jˆ7.0 Hz)
0.83 (3H, d, Jˆ6.2 Hz)
0.85 (3H, d, Jˆ6.2 Hz)
0.94 (3H, d, Jˆ7.7 Hz)

T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
2597
Figure 1. HMBC of anthrasteroids.
(CO); d 2.51 ppm (1H, d, Jˆ17.9 Hz), d 2.87 ppm (1H, d,
Jˆ17.9 Hz) and d 56.9 ppm (CH2)] in I. Finally, the stereo-
structures of I and II, including the sec-OH group at the
C-3-position, were unambiguously determined by the
synthesis of citreoanthrasteroid A and B starting from
ergosterol, as described later.
and C-14 (d 226.2 ppm) were also observed. These results
indicate that the methyl group (C-18) must be attached to
the quaternary carbon (C-13) and the CO group (C-14) to
both C-13 and C-15, respectively. The presence of a phenol
ring is suggested by the IR and 13C NMR spectral data of III
(IR 3407 cm21 and 13C NMR d 152.3 ppm). The HMBC
spectrum indicated the correlations of H-7 (d 6.51 ppm)
with C-1 (d 27.5 ppm), C-5 (d 124.8 ppm) and C-8 (d
152.3 ppm). In addition, H-19 (d 2.12 ppm, 3H) was corre-
lated with both C-9 (d 124.5 ppm) and C-10 (d 135.3 ppm).
Therefore, the phenolic ring includes C-5±C-10. In the
HMBC spectrum, correlations between H-4 (d 2.50 and
2.95 ppm, 2H) and both C-3 (d 68.2 ppm) and C-5 (d
124.8 ppm) were observed, indicating that C-4 is connected
to C-3 bearing the OH group (1H and 13C NMR d 4.10 and
68.15 ppm) and C-5 of the phenolic ring. Finally, C-1 must
be bonded to C-6 and C-11 to both C-9 and C-12.
Secocitreoanthrasteroid (III) was obtained as a colorless oil
and has a molecular formula C28H40O3. The structure of III
was determined based on the 1H and 13C NMR, HMQC and
HMBC spectra with the aid of proton homonuclear
decoupling experiments, as shown in Table 1 and Fig. 1.
The presence of one CO group is suggested by the IR and
13C NMR spectra (IR 1726 cm21 and 13C NMR d
1
226.2 ppm). The H NMR spectrum of III indicated the
presence of a trans-ole®nic double bond [d 5.25 ppm (dd,
Jˆ8.4 and 15.2 Hz, H-23) and d 5.33 ppm (dd, Jˆ7.3 and
15.2 Hz, H-22)] included in the partial structure C-20±C-28,
which is quite similar to those of citreoanthrasteroid A and
B (I and II) in the corresponding 1H and 13C NMR spectral
data. In the HMBC spectra, correlations between H-18 [d
0.93 ppm (3H, s)] and both C-12±C-14 (d 36.1, 52.5 and
226.2 ppm) and C-17 (d 46.5 ppm) were observed. In addi-
tion, correlations between H-15 (d 2.10 and 2.45 ppm, 2H)
The stereostructures of citreoanthrasteroid A and B were
unambiguously determined by synthesis starting from
ergosterol (7). The stereostructure of secocitreoanthrasteroid
has not been determined yet, but must be cited as III based
on the stereochemistry of I and II, which co-occur with
secocitreoanthrasteroid.
Scheme 3. (a) Ac2O, pyridine; (b) 4-phenyl-1,2,4-triazoline-3,5-dione, benzene; (c) BF3±OEt2, CH2Cl2, 87±95% in 3 steps; (d) PCC, Celite, benzene, 23%; (e)
K2CO3, MeOH±THF 99%; (f) NaBH4, EtOH then H2O, 88%; (g) 1 M HCl aq., Et2O±EtOH, 99%.

2598
T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
Scheme 4. (a) BzCl, pyridine, 95%; (b) NaBH4, EtOH then H2O, 99%; (c) MeI, NaH, THF, 95,96%; (d) K2CO3, MeOH±THF, 98,99%.
According to Bothworth's method,11 3-acetylergosterol was
readily converted into the corresponding anthrasteroid (8),
which was found in nature by Koshino et al.12 Further oxida-
tion of 8 with pyridinum chlorochromate (PCC) afforded
ketone 9, which was successfully hydrolyzed with K2CO3
in MeOH±THF to give rise to citreoanthrasteroid A (I). The
synthetic compound is completely identical with an authen-
tic sample of citreoanthrasteroid A in all respects of the
spectral data, including optical rotation [synthetic sample:
[a]D24ˆ29.88 (cˆ2.5 in CHCl3); natural sample: [a]2D4ˆ
27.58 (cˆ1.0 in CHCl3)] (Scheme 3).
rotation [synthetic sample: [a]2D4ˆ133.68 (cˆ1.0 in CHCl3);
natural sample: [a]D24ˆ122.78 (cˆ0.05 in CHCl3)].
In a previous paper10 we reported on the isolation and struc-
ture determination of citreoanthrasteroid (IV), which was
found in the culture ®ltrate of a hybrid strain, KO 0011,
prepared by cell fusion technique using P. citreo-viride
IFO 6200 and 4692. This metabolite was successfully
synthesized from citreoanthrsteroid A (I), as shown in
Scheme 4. Citreoanthrasteroid A (I) was treated with
benzoyl chloride in pyridine to give 12, which was reduced
with NaBH4 in EtOH to afford a mixture of two dia-
stereomers (13a and 13b). This mixture was successfully
separated by ¯ash column chromatography. Of these two
diastereomers, whose stereochemistry was determined by
NOE experiments, the a-isomer was treated with MeI and
NaH to afford methyl ether 14. Finally, 14 was hydrolyzed
with K2CO3 in MeOH±THF to give rise to citreoanthrasteroid
Furthermore, 9 was reduced with NaBH4 in MeOH to give
a mixture of two epimers, 10a and 10b, which were
directly treated with 1 M HCl to yield 11. Finally, 11 was
hydrolyzed with K2CO3 in MeOH±THF to give rise to
citreoanthrasteroid B (II), whose spectral data were com-
patible with those of an authentic sample, including optical
Scheme 5. A plausible biosynthetic pathway of citreoanthrasteroids.

T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
2599
(IV). The synthetic compound was completely identical
with an authentic sample of citreoanthrasteroid in all
respects of the spectral data (Scheme 4).
diluted overnight. A mixed solution of a high pH Ca21
solution and PS medium (5% sucrose in the potato extract)
(9:1) (100 mL) was added to the above mixture and allowed
to stand at room temperature for 10 h. Then, this mixture
was also treated with a mixed solution of a high pH Ca21
solution and PS medium (5% sucrose in the potato extract)
(3:1) (100 mL) overnight, and then 20 mL of the mixture
was suspended in 2 mL of PSA medium at 408C, sprayed
over PSA medium plates and then incubated for 7 days.
From some hybrid strains, a hopeful strain KO 0231 was
selected.
From a biogenetic point of view, in the light of Scheme 3 (7
to 8), ergosterol (7) will be oxygenated to give peroxide 16,
which is subjected to double 1,2-shifts to afford anthrasteroid
(8), as shown in Scheme 5. Then, enzymatic oxidation of 8
will afford 17, which is dehydrated or methylated to give II
or IV, respectively. Further oxidation of 17 gives rise to I.
In the present study, cell fusion technique using two
different strains has been proved to be a useful method to
®nd new bioactive compounds
Isolation of citreoanthrasteroid A (I), citreoanthra-
steroid B (II) and secocitreoanthrasteroid (III)
Polished rice (200 g) in deionized water (500 mL) was
successively entered into an Erlenmeyer ¯ask (3 L), cooked
using an autoclave (1218C, 20 min at 2 atm), and then
inoculated with a suspension of the above hybrid strain
KO 0231 in sterilized water and incubated stationary at
room temperature for 61 days, giving a yellow rice, which
was extracted with a lot of acetone. The acetone solution
was concentrated under reduced pressure to give a yellow
oil, which was partitioned between EtOAc and water. The
EtOAc extract (32 g) was chromatographed on silica gel
using a gradient solvent of EtOAc±hexane (50±100%) to
afford 6 fractions (A±F). Fraction C eluted with EtOAc
(100%) gave a brown oil, which was further separated by
repeated preparative TLC [EtOAc±hexane (1:4)] and then
EtOAc±hexane (1:4)] to afford a colorless oil including I
and II. This oil was further separated by preparative TLC
[CHCl3±MeOH (30:1)] to two fractions. The more polar
fraction was further separated by preparative TLC
[EtOAc±hexane (1:2)] to afford I as a colorless oil
(4.8 mg). The remaining fraction was subjected to HPLC
[ODS-UG-5 Develosil f10£250 mm, ¯ow 6.0 mL/min,
Rt. 9.3 min] using MeOH to afford II as a colorless oil
(0.9 mg).
Materials and Methods
General procedures
1H NMR (400 MHz) and 13C NMR spectra (100 MHz) were
recorded on a Jeol GX-400 or JNM-a400 spectrometer in
CDCl3 as solvent, unless otherwise stated. IR spectra were
measured as a ®lm on a JASCO FT/IR-410 spectrometer.
HREIMS were taken on a HITACHI M-80B GC-MS spec-
trometer. Optical rotations were determined on a JASCO
DIP-360 polarimeter. All reactions were carried out under
Ar atmosphere and monitored by TLC and reaction products
were puri®ed by using silica gel column chromatography
(Fuji Silysia BW-300).
Cell fusion
Sliced potato (400 g) was immersed in boiling water (2 L)
for 20 min and ®ltered. Each strain, P. citreo-viride IFO
6200 and 4692, was grown on PSA medium (5% sucrose
and 5% agar in the above ®ltrate) for 4 days. Spores were
separated from the mycelial debris by ®ltration through
nylon mesh and incubated on malt extract broth (3% malt
extract and 0.5% peptone in deionized water) for 12 h,
respectively. The germinal hypha were independently sepa-
rated from the malt extract broth by centrifugation
(700 rpm, 10 min), washed twice by centrifugation
(700 rpm, 10 min) with 0.1 M phosphate buffer (stabilized
with 0.6 M NaCl) and then treated with a mixed solution
[pectolyase Y23 (Seishin Pharm. Co. Ltd) (10 mg), cellulase
(Sigma C-0901) (5.0 mg), sulfatase (Sigma S-9751)
(1.10 mL) and zymolyase (Seikagaku Co. 20000 unit) in
10 mL of 0.1 M phosphate buffer (stabilized with 0.6 M
NaCl) at 308C for 3 h. Each protoplast was separated from
the mycelial debris by ®ltration through a glass ®lter
(Shibata P250) and then washed by centrifugation
(700 rpm, 5 min) with 0.1 M phosphate buffer solution
(stabilized with 0.6 M NaCl). The two different protoplasts
in a high pH Ca21 solution [(0.1 M CaCl2´2H2O and 0.8 M
mannitol in deionized water)/(0.01 M glycine in deionized
water (pH 10.5 with 2 M NaOH aq.))ˆ1:1] were mixed
(each protoplast: ca. 5£105±106 mL21). A mixed solution
of the two protoplasts (100 mL) was put on a glass plate and
suspended in a mixed solution of 50% polyethylene glycol
6000, 3.6% glucose, 0.5% CaCl2´2H2O and 0.1% KH2PO4
(100 mL) for 20 min. The suspension was diluted with a
high pH Ca21 solution (100 mL) for 20 min and then further
Fraction D eluted with EtOAc (100%) gave a brown oil,
which was further separated by repeated preparative TLC
[EtOAc±benzene (1:1) and then EtOAc±hexane (1:1)] to
afford secocitreoanthrasteroid (III) as a colorless oil
(4.1 mg).
I. [a]2D4ˆ27.58 (cˆ1.0, in CHCl3); IR: 3397, 1667, 1594,
1459, 1381, 1233 cm21; HREIMS: Found m/z 408.3026,
C28H40O2 requires m/z 408.3026. 1H and 13C NMR spectral
data are cited in Table 1.
II. [a]2D4ˆ122.78 (cˆ0.05, in CHCl3); IR: 3348 cm21
;
HREIMS: Found m/z 392.3056, C28H40O requires m/z
392.3076. 1H and 13C NMR spectral data are cited in
Table 1.
III. [a]2D4ˆ116.98 (cˆ0.5, in EtOH); IR: 3407, 1726 cm21
;
HREIMS: Found m/z 426.3153, C28H40O2 requires m/z
426.3132. 1H and 13C NMR spectral data are cited in
Table 1.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-
one-3a-acetate (9). A solution of 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-3a-acetate (8) (1.04 g, 2.4 mmol) including

2600
T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
PCC (9.56 g, 44.3 mmol), and celite (25 g) in benzene
(200 mL) was stirred for 30 h under re¯uxing, and then
cooled and ®ltered. The ®ltrate was separated by column
chromatography [Si gel, EtOAc±hexane (1:10)] to give
1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-one-3a-acetate
(9) (251 mg, 23%) as a colorless oil: [a]2D5ˆ218.1 (cˆ1.90
in CHCl3); IR: 1738, 1670, and 1595 cm21; 1H NMR d/ppm
6.71 (1H, s), 5.28±5.14 (3H, m), 3.02 (1H, dd, Jˆ5.4,
17.1 Hz), 2.93±2.83 (4H, m), 2.70 (1H, dd, Jˆ6.4,
17.1 Hz), 2.53 (1H, d, Jˆ7.6 Hz), 2.45 (3H, s), 2.07±1.83
(6H, m), 2.03 (3H, s), 1.63 (1H, dq, Jˆ6.4, 12.2 Hz), 1.55±
1.43 (3H, m), 1.03 (3H, d, Jˆ6.4 Hz), 0.91 (3H, d,
Jˆ6.8 Hz), 0.83 (3H, d, Jˆ6.8 Hz), 0.81 (3H, d,
Jˆ6.8 Hz), 061 (3H, s); 13C NMR d/ppm 201.2, 170.8,
142.8, 140.5, 139.6, 134.9, 132.7, 131.7, 129.2, 124.2,
68.9, 56.9, 55.9, 50.7, 44.4, 42.9, 40.5, 33.2, 32.5, 28.8,
27.4, 26.9, 24.1, 21.5, 20.7, 20.1, 19.8, 17.7, 16.5, 13.4;
HREIMS: Found m/z 450.3161, C30H42O3 requires m/z
450.3131.
1H NMR (400 MHz, C6D6) d/ppm 6.70 (1H, s), 5.32±5.21
(2H, m), 4.84 (1H, d, Jˆ5.2 Hz), 2.89 (1H, dd, Jˆ5.2,
16.8 Hz), 2.74 (1H, dt, Jˆ5.6, 16.4 Hz), 2.63±2.57 (2H,
m), 2.45±2.42 (2H, m), 2.13 (3H, s), 2.13±2.09 (1H, m),
2.02±1.78 (4H, m), 1.72 (3H, s), 1.71±1.64 (2H, m), 1.55±
1.47 (3H, m), 1.35±1.20 (1H, m), 1.17 (3H, d, Jˆ6.4 Hz),
1.02 (3H, d, Jˆ6.8 Hz), 0.92 (3H, d, Jˆ6.4 Hz), 0.91 (3H, d,
Jˆ6.8 Hz), 0.86 (3H, s); 13C NMR (100 MHz, C6D6) d/ppm
169.7, 137.1, 136.8, 136.1, 134.8, 134.4, 132.4, 130.9,
124.0, 70.8, 66.8, 55.6, 53.2, 48.4, 43.5, 41.2, 40.9, 33.7,
33.4, 29.6, 28.2, 27.8, 24.7, 21.4, 21.2, 20.4, 20.2, 18.2,
15.9, 13.6; HREIMS: Found m/z 452.2390, C30H44O3
requires m/z 452.2388.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,11,22-hexaen-
3a-acetate (11). A solution of 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-11-hydroxy-3a-acetate (10) (52.4 mg,
0.12 mol) in Et2O (3 mL) was poured into a mixed solution
of 1 M HCl aq. (2 mL) and EtOH (3 mL) and then stirred at
room temperature for 5 h. The reaction mixture was poured
into saturated aqueous solution NaHCO3 (5 mL) and then
extracted with EtOAc (3£20 ml) at 08C. The combined
extracts were dried over Na2SO4 and concentrated to give
a crude oil, which was then puri®ed by column chromato-
graphy [Si gel, EtOAc±hexane (1:4)] to afford
1(10!6)abeo-ergosta-5,7,9,11,22-hexaen-3a-acetate (11)
(49.9 mg, 99%) as a colorless oil: [a]2D5ˆ135.38 (cˆ1.00
Synthesis of citreoanthrasteroid A (I). A solution of
1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-one-3a-acetate
(9) (92 mg, 0.21 mmol) and K2CO3 (1.5 g, 1.1 mmol) in a
mixed solvent of MeOH (10 mL) and THF (3 mL) was stir-
red at room temperature for 30 min, and ®ltered through
Celite. The ®ltrate was poured into water and extracted
with EtOAc (3£10 mL). The combined extracts were
dried over Na2SO4 and concentrated to give a crude product,
which was then separated by column chromatography [Si
gel, EtOAc±hexane (1:4)] to afford citreoanthrasteroid A (I) as
a colorless oil (84 mg, 99%): [a]2D4ˆ29.88 (cˆ2.5, in CHCl3).
1
in CHCl3); IR: 1738 cm21; H NMR d/ppm 6.55 (1H, s),
6.47 (1H, d, Jˆ10.0 Hz), 6.39 (1H, d, Jˆ10.0 Hz), 5.20±
5.09 (3H, m), 2.93 (1H, dd, Jˆ5.4, 16.6 Hz), 2.83±2.72 (3H,
m), 2.55 (1H, dd, Jˆ6.8, 17.1 Hz), 2.07 (3H, s), 1.95 (3H, s),
1.95±1.75 (6H, m), 1.59 (1H, q, Jˆ9.3 Hz), 1.52 (1H, dd,
Jˆ5.9, 12.2 Hz), 1.41±1.35 (2H, m), 1.04 (3H, d,
Jˆ6.3 Hz), 0.83 (3H, d, Jˆ6.8 Hz), 0.75 (3H, d,
Jˆ6.8 Hz), 0.73 (3H, d, Jˆ6.3 Hz), 0.49 (3H, s); 13C
NMR d/ppm 170.9, 140.4, 140.1, 137.3, 135.2, 134.0,
132.5, 131.8, 130.9, 129.6, 123.6, 123.1, 70.6, 51.8, 50.3,
43.6, 42.9, 41.0, 33.2, 32.8, 29.6, 27.5, 27.3, 22.0, 20.9,
20.1, 19.8, 17.8, 14.5, 11.6; HREIMS: Found m/z
434.3161, C30H42O3 requires m/z 434.3181.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-
hydroxy-3a-acetate (10a and 10b). A solution of
1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-one-3a-acetate
(9) (133 mg, 0.30 mol) and NaBH4 (66.7 mg, 1.7 mmol) in
EtOH (10 ml) was stirred for 5 min at 08C, and then poured
into water (50 mL) and allowed to stand at room tempera-
ture for 1 h. The reaction mixture was extracted with EtOAc
(3£25 ml). The combined extracts were washed with
saturated NaCl aq. (20 mL), dried over Na2SO4, and concen-
trated to give a crude oil, which was then separated by
column chromatography [Si gel, EtOAc±hexane (1:5)] to
afford a diastereomeric mixture (120 mg, 88%) as a color-
less oil. This mixture was separated by ¯ash column
chromatography [Si gel, acetone±hexane (1:10)] to afford
1(10!6)abeo-ergost-5,7,9,22-tetraen-11a-hydroxy-3a-
acetate (10a) as a colorless oil (43.8 mg): [a]2D6ˆ228.08
Synthesis of citreoanthrasteroid B (II). A solution of
1(10!6)abeo-ergosta-5,7,9,11,22-hexaen-3a-acetate (11)
(49.9 mg, 0.11 mmol) and K2CO3 (0.50 g, 3.6 mmol) in a
mixed solvent of MeOH (5 mL) and THF (1 mL) was stirred
at room temperature for 6 h, and then ®ltered through Celite.
The ®ltrate was poured into water and extracted with EtOAc
(3£10 mL). The combined extracts were dried over Na2SO4
and concentrated to give a crude product, which was then
separated by column chromatography [Si gel, EtOAc±
hexane (1:4)] to give citreoanthrasteroid B (II) (44.6 mg,
99%) as a colorless oil: [a]2D4ˆ133.68 (cˆ1.0 in CHCl3).
The spectral data of the synthetic compound were com-
pletely identical with those of an authentic sample.
1
(cˆ1.00 in CHCl3); IR: 3276 and 1738 cm21; H NMR
(400 MHz, C6D6) d/ppm 6.69 (1H, s), 5.37±5.30 (3H, m),
5.05 (1H, t, Jˆ7.6 Hz), 2.87±2.76 (3H, m), 2.66±2.54 (3H,
m), 2.17 (3H, s), 2.01±1.25 (11H, m), 1.69 (3H, s), 1.11 (3H,
d, Jˆ6.4 Hz), 1.01 (3H, d, Jˆ6.8 Hz), 0.92 (3H, d,
Jˆ6.8 Hz), 0.91 (3H, d, Jˆ6.8 Hz), 0.46 (3H, s); 13C
NMR (100 MHz, C6D6) d/ppm 169.8, 138.3, 136.2, 136.0,
134.8, 134.5, 132.4, 130.7, 123.9, 70.4, 67.3, 56.0, 51.3,
49.8, 45.4, 43.5, 41.2, 33.7, 33.2, 29.6, 27.8, 27.3, 24.8,
21.1, 20.5, 20.1, 18.2, 15.6, 13.0; HREIMS: Found m/z
452.2354, C30H44O3 requires m/z 452.2388.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-
one-3a-benzoate (12). A solution of citreoanthrasteroid A
(I) (252 mg 0.62 mmol) and benzoyl chloride (236 mg,
1.7 mmol) in pyridine (10 mL) was stirred at 08C for 5 h.
The reaction mixture was poured into saturated aqueous
solution NaHCO3 (10 mL). The mixture was extracted
with EtOAc (3£30 mL). The combined extracts were
dried over Na2SO4 and concentrated to give a crude product,
1(10!6)abeo-ergosta-5,7,9,22-tetraen-11b-hydroxy-3a-
acetate (10b) (38.9 mg) was also obtained as a colorless oil:
[a]2D6ˆ11.808 (cˆ2.10 in CHCl3). IR: 3415 and 1738 cm21
;

T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
2601
which was then separated by column chromatography [Si
gel, EtOAc±hexane (1:8)] to afford 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-11-one-3a-benzoate (302 mg, 95%) as a
colorless oil: [a]2D5ˆ111.88 (cˆ2.50 in CHCl3); IR: 1717,
1670 and 1596 cm21; 1H NMR d/ppm 8.00 (2H, dd, Jˆ1.5,
7.8 Hz), 7.53 (1H, tt, Jˆ1.5, 7.8 Hz), 7.40 (2H, t, Jˆ7.8 Hz),
6.75 (1H, s), 5.53 (1H, quintet, Jˆ5.9 Hz), 5.25 (1H, dd,
Jˆ7.3, 15.1 Hz), 5.18 (1H, dd, Jˆ8.3, 15.1 Hz), 3.14 (1H,
dd, Jˆ5.4, 16.6 Hz), 3.04 (1H, dt, Jˆ6.8, 17.6 Hz), 2.94±
2.84 (4H, m), 2.54 (1H, d, Jˆ18.1 Hz), 2.48 (3H, s), 2.12±
2.03 (4H, m), 2.00±1.91 (1H, m), 1.87 (1H, q, Jˆ6.8 Hz),
1.65 (1H, dq, Jˆ5.4, 6.4 Hz), 1.55±1.44 (3H, m), 1.01 (3H,
d, Jˆ6.4 Hz), 0.92 (3H, d, Jˆ6.8 Hz), 0.84 (3H, d,
Jˆ6.8 Hz), 0.82 (3H, d, Jˆ6.3 Hz), 0.63 (3H, s,); 13C
NMR d/ppm 201.0, 165.9, 142.6, 140.4, 139.5, 134.7,
132.8, 132.5, 131.6, 130.3, 129.5, 129.1, 128.2, 124.1,
70.3, 56.8, 55.9, 50.7, 44.4, 42.9, 40.4, 33.1, 32.6, 28.8,
27.4, 27.0, 24.0, 20.7, 20.0, 19.8, 17.7, 16.5, 13.4; HREIMS:
Found m/z 512.3266, C30H42O3 requires m/z 512.3245.
(5H, m), 1.74 (1H, dd, Jˆ5.4, 13.7 Hz), 1.64 (1H, dd,
Jˆ8.3, 13.7 Hz), 1.61±1.53 (2H, m), 1.29±1.26 (2H, m),
1.22 (3H, d, Jˆ6.8 Hz), 1.07 (3H, d, Jˆ6.8 Hz), 0.978
(3H, d, Jˆ6.8 Hz), 0.982 (3H, d, Jˆ6.8 Hz), 0.92 (3H, s);
13C NMR (100 MHz, C6D6): d/ppm 165.8, 137.1, 136.9,
136.1, 134.8, 134.4, 132.8, 132.4, 131.4, 130.8, 129.9,
128.5, 124.1, 71.4, 66.8, 55.6, 53.2, 48.4, 43.5, 41.2, 40.9,
33.6, 33.3, 29.6, 28.1, 27.6, 24.7, 21.5, 20.5, 20.2, 18.2,
16.0, 13.6; HREIMS: Found m/z 514.3425, C35H46O3
requires m/z 514.3444.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11a-
methoxy-3a-benzoate (14a). A solution of 1(10!6)abeo-
ergosta-5,7,9,22-tetraen-11a-hydroxy-3a-benzoate (95 mg,
0.18 mmol) and MeI (1.2 mL, 12.7 mmol) in THF (10 mL)
was stirred at 2788C under Ar. Then, NaH (24.5 mg (60%),
0.61 mmol) was added to the solution at 2788C and stirred
at the same temperature for 10 min under Ar. The reaction
solution was poured into saturated NH4Cl aq. (10 mL) at
08C. The mixture was extracted with EtOAc (3£30 mL).
The combined extracts were dried over Na2SO4 and then
concentrated to give a crude oil, which was separated by
column chromatography [Si gel, EtOAc±benzene (20:1)] to
afford 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11a-methoxy-
3a-benzoate (14a) (93 mg, 95%) as a colorless oil:
[a]2D5ˆ21.28 (cˆ2.21 in CHCl3); IR: 1715 and
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-
hydroxy-3a-benzoate (13a and 13b). A solution of
1(10!6)abeo-ergosta-5,7,9,22-tetraen-11-one-3a-benzoate
(12) (302 mg, 0.59 mol) and NaBH4 (126 mg, 3.3 mmol) in
EtOH (20 mL) was stirred at 08C for 5 min. The reaction
mixture was poured into water (60 mL) and allowed to stand
at room temperature for 1 h. The mixture was extracted with
EtOAc (3£25 mL). The combined extracts were washed
with saturated NaCl aq. (20 mL) and then dried over
Na2SO4 and concentrated to give a crude oil, which was
separated by column chromatography [Si gel, EtOAc±
hexane (1:5)] to give 1(10!6)abeo-ergosta-5,7,9,22-
tetraen-11-hydroxy-3a-benzoate (302 mg, 99%) as a dia-
stereomeric mixture. This mixture was successfully
separated by ¯ash column chromatography [Si gel,
acetone±hexane (1:10)] to give 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-11a-hydroxy-3a-benzoate (13a) (119 mg)
as a colorless oil: [a]D26ˆ220.38 (cˆ1.19 in CHCl3); IR:
1
1603 cm21; H NMR (400 MHz, C6D6): d/ppm 8.16 (2H,
dd, Jˆ1.6, 7.2 Hz), 7.09 (1H, dt, Jˆ1.6, 7.2 Hz), 7.01 (2H, t,
Jˆ7.2 Hz), 6.75 (1H, s), 5.58 (1H, m), 5.30 (1H, dd, Jˆ7.2,
15.2 Hz), 5.23 (1H, dd, Jˆ7.6, 15.2 Hz), 4.74 (1H, dd,
Jˆ6.0, 8.0 Hz), 3.10 (3H, s), 3.06±2.97 (2H, m), 2.82±
2.77 (2H, m), 2.65(1H, m), 2.43 (1H, dd, Jˆ7.6, 13.2 Hz),
2.15 (3H, s), 2.07±2.04 (1H, m), 1.97± 1.83 (6H, m), 1.64±
1.55 (1H, m), 1.51 (1H, q, Jˆ6.8 Hz), 1.44±1.39 (2H, m),
1.13 (3H, d, Jˆ6.8 Hz), 1.03 (3H, d, Jˆ6.8 Hz), 0.94 (3H, d,
Jˆ6.8 Hz), 0.93 (3H, d, Jˆ6,0 Hz), 0.50 (3H, s); 13C NMR
(100 MHz, C6D6): d/ppm 165.8, 139.4, 136.6, 136.0, 134.9,
132.7, 132.4, 132.0, 131.4, 130.2, 129.9, 128.5, 123.8, 75.2,
71.2, 56.2, 53.8, 50.6, 45.3, 44.9, 43.5, 41.2, 33.6, 33.3,
29.7, 27.5, 24.4, 21.4, 20.4, 20.2, 18.2, 15.3, 14.0; HREIMS:
Found m/z 528.3593, C36H48O3 requires m/z 528.3600.
1
3281, 1717 cm21; H NMR (400 MHz, C6D6): d/ppm 8.22
(2H, dd, Jˆ1.5, 7.3 Hz), 7.13 (1H, t, Jˆ7.3 Hz), 7.05 (2H, t,
Jˆ7.3 Hz), 6.77 (1H, s), 5.58 (1H, m), 5.34 (1H, dd, Jˆ8.3,
15.1 Hz), 5.32 (1H, dd, Jˆ7.8, 15.1 Hz), 5.03 (1H, dd,
Jˆ7.7, 7.8 Hz), 2.93±2.89 (3H, m), 2.80 (1H, dd, Jˆ5.9,
7.1 Hz), 2.68±2.60 (2H, m), 2.23 (3H, s), 2.07±1.95 (5H,
m), 1.87 (1H, m), 1.69±1.40 (7H, m), 1.17 (3H, d,
Jˆ6.4 Hz), 1.07 (3H, d, Jˆ6.8 Hz), 0.978 (3H, d,
Jˆ6.8 Hz), 0.982 (3H, d, Jˆ6.3 Hz), 0.53 (3H, s); 13C
NMR (100 MHz, C6D6): d/ppm 165.9, 138.3, 136.4,
136.0, 134.8, 134.5, 132.8, 132.4, 131.3, 130.6, 129.9,
128.5, 123.9, 71.1, 67.2, 56.0, 51.4, 49.8, 45.4, 43.5, 41.2,
33.6, 33.2, 29.6, 27.8, 27.2, 24.8, 21.4, 20.5, 20.2, 18.2,
15.6, 12.9; HREIMS: Found m/z 514.3488, C35H46O3
requires m/z 514.3444. 1(10!6)abeo-ergosta-5,7,9,22-
tetraen-11b-hydroxy-3a-benzoate (13b) (115 mg) was
also obtained as a colorless oil: [a]2D5ˆ115.48 (cˆ2.00 in
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11b-
methoxy-3a-benzoate (14b). A solution of 1(10!6)abeo-
ergosta-5,7,9,22-tetraen-11b-hydroxy-3a-benzoate (87 mg,
0.18 mmol) and MeI (1.2 mL, 12.7 mmol) in THF (10 mL)
was stirred at 2788C under Ar. Then, NaH (24.5 mg (60%),
0.61 mmol) was added to the solution at 2788C and stirred
at same temperature for 10 min under Ar. The mixture was
poured into saturated NH4Cl aq. (10 mL) at 08C. The reac-
tion mixture was extracted with EtOAc (3£30 mL). The
combined extracts were dried over Na2SO4 and concen-
trated to give a crude oil, which was then separated by
column chromatography [Si gel, EtOAc±benzene (20:1)]
to give 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11b-methoxy-
3a-benzoate (14b) (86 mg, 96%) as a colorless oil:
[a]2D5ˆ20.48 (cˆ2.75 in CHCl3); IR: 1718 and
1
CHCl3); IR: 3422 and 1717 cm21; H NMR (400 MHz,
C6D6): d/ppm 8.24 (2H, dd, Jˆ1.5, 6.8 Hz), 7.15 (1H, dt,
Jˆ1.5, 6.8 Hz), 7.08 (2H, d, Jˆ6.8 Hz), 6.79 (1H, s), 5.56
(1H, m), 5.35 (1H, dd, Jˆ7.3, 15.1 Hz), 5.29 (1H, dd,
Jˆ6.8, 15.1 Hz), 4.90 (1H, br.s), 2.99 (1H, dd, Jˆ4.9,
16.6 Hz), 3.88 (1H, dt, Jˆ5.9, 16.6 Hz), 2.78 (1H, dd,
Jˆ6.3, 16.6 Hz), 2.69 (1H, dt, Jˆ6.3, 17.6 Hz), 2.50 (1H,
d, Jˆ13.2 Hz), 2.51±2.48 (1H, m), 2.18 (3H, s), 2.06±1.94
1
1602 cm21; H NMR (400 MHz, C6D6): d/ppm 8.17 (2H,
dd, Jˆ0.8, 8.4 Hz), 7.09 (1H, dt, Jˆ0.8, 8.4 Hz), 7.02 (2H, t,
Jˆ8.4 Hz), 6.76 (1H, s), 5.49 (1H, m), 5.32 (1H, dd, Jˆ4.4,
15.3 Hz), 5.27 (1H, dd, Jˆ5.2, 15.3 Hz), 4.19 (1H, d,
Jˆ4.8 Hz), 3.19 (3H, s), 2.93 (1H, dd, Jˆ5.4, 17.1 Hz),
2.88±2.78 (2H, m), 2.65 (1H, d, Jˆ13.7 Hz), 2.67±2.59

2602
T. Nakada, S. Yamamura / Tetrahedron 56 (2000) 2595±2602
1
(1H, m), 2.54 (1H, dd, Jˆ7.8, 12.2 Hz), 2.15 (1H, dt, Jˆ6.8,
10.3 Hz), 2.07 (3H, s), 2.07±2.00 (1H, m), 1.98±1.85 (4H,
m), 1.64 (1H, dq, Jˆ5.7, 6.8 Hz), 1.55±1.47 (2H, m), 1.39
(1H, dd, Jˆ5.4, 14.2 Hz), 1.29 (1H, q, Jˆ7.8 Hz), 1.23 (3H,
d, Jˆ6.8 Hz), 1.03 (3H, d, Jˆ6.8 Hz), 0.94 (3H, s), 0.93
(6H, d, Jˆ7.8 Hz); 13C NMR (100 MHz, C6D6): d/ppm
165.7, 137.6, 137.3, 136.1, 134.9, 133.1, 132.7, 132.4,
131.5, 130.4, 129.9, 128.5, 123.9, 76.4, 71.3, 55.6, 55.5,
53.3, 43.5, 41.2, 40.6, 33.6, 33.4, 29.8, 28.0, 27.5, 24.8,
21.5, 20.4, 20.2, 18.2, 15.9, 12.9; HREIMS: Found m/z
528.3592, C36H48O3 requires m/z 528.3600.
and 1602 cm21; H NMR (400 MHz, C6D6): d/ppm 6.74
(1H, s), 5.31 (1H, dd, Jˆ2.9, 15.1 Hz), 5.27 (1H, dd,
Jˆ3.4, 15.1 Hz), 4.20 (1H, d, Jˆ4.9 Hz), 3.61 (1H, m),
3.20 (3H, s), 2.82 (1H, dd, Jˆ5.4, 16.6 Hz), 2.77 (1H, dt,
Jˆ4.9, 17.6 Hz), 2.64 (1H, d, Jˆ14.2 Hz), 2.62±2.58 (1H,
m), 2.53 (1H, dd, Jˆ7.3, 11.2 Hz), 2.45 (1H, dd, Jˆ8.3,
16.1 Hz), 2.13 (3H, s), 2.13 (1H, m), 2.03±1.91 (3H, m),
1.75 (1H, m), 1.64±1.47 (4H, m), 1.39 (1H, dd, Jˆ4.9,
14.2 Hz), 1.28 (1H, dq, Jˆ3.4, 10.7 Hz), 1.23 (3H, d,
Jˆ6.3 Hz), 1.02 (3H, d, Jˆ6.8 Hz), 0.93 (6H, d,
Jˆ7.8 Hz), 0.91 (3H, s); 13C NMR (100 MHz, C6D6):
d/ppm 137.5, 137.1, 136.1, 135.3, 132.9, 132.4, 131.7,
123.8, 76.5, 67.9, 55.6, 53.3, 43.5, 41.20, 41.18, 40.6,
37.4, 33.6, 32.1, 29.8, 28.8, 24.8, 21.5, 20.4, 20.2, 18.2,
16.2, 12.9; HREIMS: Found m/z 424.3371, C29H44O2
requires m/z 424.3339.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11a-
methoxy-3a-ol (IV). A solution of 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-11a-methoxy-3a-benzoate (14a) (95 mg,
0.18 mmol) and K2CO3 (1.0 g, 0.73 mmol) in a mixed
solvent of MeOH (5 mL) and THF (1.5 mL) was stirred at
room temperature for 30 min and then ®ltered through
Celite. The ®ltrate was poured into water and extracted
with EtOAc (3£10 mL). The combined extracts were
dried over Na2SO4 and concentrated to give a crude product,
which was then separated by column chromatography [Si
gel, EtOAc±hexane (1:4)] to give citreoanthrasteroid (IV)10
(75 mg, 98%) as a colorless oil: [a]2D5ˆ2188 (cˆ1.35 in
Acknowledgements
The authors are indebted to the Ministry of Education,
Science, Sports and Culture (Japan) for Grant-in-Aid for
Special Promotion Research No. 09101001.
1
CHCl3); IR: 3386 and 1604 cm21; H NMR (400 MHz,
C6D6), d/ppm 6.74 (1H, s), 5.28 (1H, dd, Jˆ5.4, 15.6 Hz),
5.23 (1H, dd, Jˆ5.9, 15.6 Hz), 4.77 (1H, dd, Jˆ7.3, 7.8 Hz),
3.81 (1H, m), 3.12 (3H, s), 3.05 (1H, dd, Jˆ6.3, 10.7 Hz),
2.86±2.77 (2H, m), 2.61 (1H, dt, Jˆ6.8, 8.3 Hz), 2.45± 2.37
(2H, m), 2.22 (3H, s), 2.03 (1H, m), 1.96±1.88 (4H, m), 1.72
(1H, m), 1.60±1.46 (3H, m), 1.43±1.40 (2H, m), 1.13 (3H,
d, Jˆ7.2 Hz), 1.01 (3H, d, Jˆ7.2 Hz), 0.92 (3H, d,
Jˆ6.8 Hz), 0.471 (3H, s); 13C NMR (100 MHz, C6D6):
d/ppm 139.2, 136.4, 136.0, 135.3, 132.4, 131.7, 131.2,
123.8, 75.2, 67.8, 56.2, 53.8, 50.6, 45.3, 44.9, 43.5, 41.2,
37.1, 33.6, 31.6, 29.7, 28.2, 24.5, 21.3, 20.4, 20.1, 18.2,
15.4, 14.0; HREIMS: Found m/z 424.3305, C29H44O2
requires m/z 424.3339.
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8. Kosemura, S.; Miyata, H.; Yamamura, S.; Albone, K.; Simpson,
T. J. J. Chem. Soc. Perkin Trans. 1 1994, 135.
Synthesis of 1(10!6)abeo-ergosta-5,7,9,22-tetraen-11b-
methoxy-3a-ol. (15). A solution of 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-11b-methoxy-3a-benzoate (14b) (82 mg,
0.16 mmol) and K2CO3 (1.0 g, 0.73 mmol) in a mixed
solvent of MeOH (5 mL) and THF (1.5 mL) was stirred
for 30 min, and then ®ltered through Celite. The ®ltrate
was poured into water and extracted with EtOAc
(3£10 mL). The combined extracts were dried over
Na2SO4 and concentrated to give a crude product, which
was then separated by column chromatography [Si gel,
EtOAc±hexane (1:4)] to afford 1(10!6)abeo-ergosta-
5,7,9,22-tetraen-11b-methoxy-3a-ol (15) (66 mg, 99%) as
a colorless oil: [a]2D5ˆ113.38 (cˆ1.00 in CHCl3) IR: 3399
9. Nakada, T.; Sudo, S.; Kosemura, S.; Yamamura, S. Tetrahedron
Lett. 1999, 40, 6831.
10. Kosemura, S.; Uotsu, S.; Yamamura, S. Tetrahedron Lett.
1997, 38, 7373.
11. Bothworth, N.; Emke, A.; Midgley, J. M.; Moore, C. J.;
Whalley, W. B.; Ferguson, G.; Marsh, C. J. Chem. Soc. Perkin
Trans. 1 1977, 805.
12. Koshino, H.; Yoshihara, T.; Sakamura, S.; Shimanuki, T.;
Sato, T.; Tajimi, A. Phytochemistry 1997, 28, 771.